The present invention provides novel pharmaceuticals useful for the diagnosis and treatment of cancer, methods of imaging tumors in a patient, and methods of treating cancer in a patient. The pharmaceuticals are comprised of a targeting moiety that binds to the vitronectin receptor that is expressed in tumor vasculature, an optional linking group, and a therapeutically effective radioisotope or diagnostically effective imageable moiety. The therapeutically effective radioisotope emits a gamma ray or alpha particle sufficient to be cytotoxic. The imageable moiety is a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.
Cancer is a major public health concern in the United States and around the world. It is estimated that over 1 million new cases of invasive cancer will be diagnosed in the United States in 1998. The most prevalent forms of the disease are solid tumors of the lung, breast, prostate, colon and rectum. Cancer is typically diagnosed by a combination of in vitro tests and imaging procedures. The imaging procedures include X-ray computed tomography, magnetic resonance imaging, ultrasound imaging and radionuclide scintigraphy. Frequently, a contrast agent is administered to the patient to enhance the image obtained by X-ray CT, MRI and ultrasound, and the administration of a radiopharmaceutical that localizes in tumors is required for radionuclide scintigraphy.
Treatment of cancer typically involves the use of external beam radiation therapy and chemotherapy, either alone or in combination, depending on the type and extent of the disease. A number of chemotherapeutic agents are available, but generally they all suffer from a lack of specificity for tumors versus normal tissues, resulting in considerable side-effects. The effectiveness of these treatment modalities is also limited, as evidenced by the high mortality rates for a number of cancer types, especially the more prevalent solid tumor diseases. More effective and specific treatment means continue to be needed.
Despite the variety of imaging procedures available for the diagnosis of cancer, there remains a need for improved methods. In particular, methods that can better differentiate between cancer and other pathologic conditions or benign physiologic abnormalities are needed. One means of achieving this desired improvement would be to administer to the patient a metallopharmaceutical that localizes specifically in the tumor by binding to a receptor expressed only in tumors or expressed to a significantly greater extent in tumors than in other tissue. The location of the metallopharmaceutical could then be detected externally either by its imageable emission in the case of certain radiopharmaceuticals or by its effect on the relaxation rate of water in the immediate vicinity in the case of magnetic resonance imaging contrast agents.
This tumor specific metallopharmaceutical approach can also be used for the treatment of cancer when the metallopharmaceutical is comprised of a particle emitting radioisotope. The radioactive decay of the isotope at the site of the tumor results in sufficient ionizing radiation to be toxic to the tumor cells. The specificity of this approach for tumors minimizes the amount of normal tissue that is exposed to the cytotoxic agent and thus may provide more effective treatment with fewer side-effects.
Previous efforts to achieve these desired improvements in cancer imaging and treatment have centered on the use of radionuclide labeled monoclonal antibodies, antibody fragments and other proteins or polypeptides that bind to tumor cell surface receptors. The specificity of these radiopharmaceuticals is frequently very high, but they suffer from several disadvantages. First, because of their high molecular weight, they are generally cleared from the blood stream very slowly, resulting in a prolonged blood background in the images. Also, due to their molecular weight they do not extravasate readily at the site of the tumor and then only slowly diffuse through the extravascular space to the tumor cell surface. This results in a very limited amount of the radiopharmaceutical reaching the receptors and thus very low signal intensity in imaging and insufficient cytotoxic effect for treatment.
Alternative approaches to cancer imaging and therapy have involved the use of small molecules, such as peptides, that bind to tumor cell surface receptors. An In-111 labeled somatostatin receptor binding peptide, In-111-DTPA-D-Phe1-octeotide, is in clinical use in many countries for imaging tumors that express the somatostatin receptor (Baker, et al. Life Sci., 1991, 49, 1583-91 and Krenning, et al., Eur. J. Nucl. Med., 1993, 20, 716-31). Higher doses of this radiopharmaceutical have been investigated for potential treatment of these types of cancer (Krenning, et al., Digestion, 1996, 57, 57-61). Several groups are investigating the use of Tc-99m labeled ananlogs of In-111-DTPA-D-Phel1-octeotide for imaging and Re-186 labeled analogs for therapy (Flanagan, et al., U.S. Pat. No. 5,556,939, Lyle, et al., U.S. Pat. No. 5,382,654, and Albert et al., U.S. Pat. No. 5,650,134).
Angiogenesis is the process by which new blood vessels are formed from pre-existing capillaries or post capillary venules; it plays a key role in the pathological development of many solid tumor cancers and their metastases. Tumor released cytokines or angiogenic factors stimulate vascular endothelial cells by interacting with specific cell surface receptors for the factors. The endothelial cells then proliferate and invade into the tumor tissue. The endothelial cells differentiate to form lumens, making new vessel offshoots of pre-existing vessels. The new blood vessels then provide nutrients to the tumor permitting further growth and a route for metastasis.
Angiogenesis is also influenced by cell adhesion molecules (Folkman, J., Nature Medicine, 1995, 1, 27-31). The integrin xcex1vxcex23 is a receptor for a wide variety of extracellular matrix proteins with an exposed tripeptide Arg-Gly-Asp moiety and mediates cellular adhesion to its ligands: vitronectin, fibronectin, and fibrinogen, among others. The integrin xcex1vxcex23 is minimally expressed on normal blood vessels, but, is significantly upregulated on vascular cells within a variety of human tumors. The role of the xcex1vxcex23 receptors is to mediate the interaction of the endothelial cells and the extracellular matrix and facilitate the migration of the cells in the direction of the angiogenic signal, the tumor cell population.
Because of the importance of angiogenesis to tumor growth and metastasis, a number of chemotherapeutic approaches are being developed to interfere with or prevent this process. One of these approaches, involves the use of anti-angiogenic proteins such as angiostatin and endostatin. Angiostatin is a 38 kDa fragment of plasminogen that has been shown in animal models to be a potent inhibitor of endothelial cell proliferation. (O""Reilly et. al. , Cell, 1994, 79, 315-328) Endostatin is a 20 kDa C-terminal fragment of collagen XVIII that has also been shown to be a potent inhibitor. (O""Reilly et. al., Cell, 1997, 88, 277-285) Systemic therapy with endostatin has been shown to result in strong anti-tumor activity in animal models. However, human clinical trials of these two chemotherapeutic agents of biological origin have been hampered by lack of availability.
Another approach to anti-angiogenic therapy is to use targeting moieties that interact with endothelial cell surface receptors expressed in the angiogenic vasculature to which are attached chemotherapeutic agents. Burrows and Thorpe (Proc. Nat. Acad. Sci, USA, 1993, 90, 8996-9000) described the use of an antibody-immunotoxin conjugate to eradicate tumors in a mouse model by destroying the tumor vasculature. The antibody was raised against an endothelial cell class II antigen of the major histocompatibility complex and was then conjugated with the cytotoxic agent, deglycosylated ricin A chain. The same group (Clin. Can. Res., 1995, 1, 1623-1634) investigated the use of antibodies raised against the endothelial cell surface receptor, endoglin, conjugated to deglycosylated ricin A chain. Both of these conjugates exhibited potent anti-tumor activity in mouse models. However, both still suffer drawbacks to routine human use. As with most antibodies or other large, foreign proteins, there is considerable risk of immunologic toxicity which could limit or preclude administration to humans. Also, while the vasculature targeting may improve the local concentration of the attached chemotherapeutic agents, the agents still must be cleaved from the antibody carrier and be transported or diffuse into the cells to be cytotoxic.
Thus, it is desirable to provide anti-angiogenic pharmaceuticals and tumor or new vasculature imaging agents which do not suffer from poor diffusion or transportation, possible immunologic toxicity, limited availability, and/or a lack of specificity.
There is also a growing interest in therapeutic angiogenesis to improve blood flow in regions of the body that have become ischemic or poorly perfused. Several investigators are using growth factors administered locally to cause new vasculature to form either in the limbs or the heart. The growth factors VEGF and bFGF are the most common for this application. Recent publications include: Takeshita, S., et. al., J. Clin. Invest., 1994, 93, 662-670; and Schaper, W. and Schaper, J., Collateral Circulation:Heart, Brain, Kidney, Limbs, Kluwer Academic Publishers, Boston, 1993. The main applications that are under investigation in a number of laboratories are for improving cardiac blood flow and in improving peripheral vessal blood flow in the limbs. For example, Henry, T. et. al. (J. Amer. College Cardiology, 1998, 31, 65A) describe the use of recombinant human VEGF in patients for improving myocardial perfusion by therapeutic angiogenesis. Patients received infusions of rhVEGF and were monitored by nuclear perfusion imaging 30 and 60 days post treatment to determine improvement in myocardial perfusion. About 50% of patients showed improvement by nuclear perfusion imaging whereas 5/7 showed new collatoralization by angiography.
Thus, it is desirable to discover a method of monitoring improved cardiac blood flow which is targeted to new collateral vessels themselves and not, as in nuclear perfusion imaging, a regional consequence of new collatoral vessels.
Another therapeutic application of the radiopharmaceuticals of the present invention that emit cytotoxic radiation (Beta and Alpha particles and Auger electons) is in treating rheumatoid arthritis (RA). In RA, the ingrowth of a highly vascularized pannus is caused by the excessive production of angiogenic factors by the infiltrating macrophages, immune cells, or inflammatory cells. Therefore, the radiopharmaceuticals of the present inventions can be used to destroy the new angiogenic vasculature that results and thus treat the disease.
It is one object of the present invention to provide improved anti-angiogenic pharmaceuticals, comprised of a targeting moiety that binds to the vitronectin receptor that is expressed in tumor neovasculature, an optional linking group, and a radioisotope. The vitronectin receptor binding compounds target the radioisotope to the tumor neovasculature. The beta or alpha-particle emitting radioisotope emits a cytotoxic amount of ionizing radiation which results in cell death. The penetrating ability of radiation obviates the requirement that the cytotoxic agent diffuse or be transported into the cell to be cytotoxic.
It is another object of the present invention to provide tumor imaging agents, comprised of tumor neovasculature vitronectin receptor binding compounds conjugated to an imageable moiety, such as a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.
It is one object of the present invention to provide anti-angiogenic pharmaceuticals, comprised of a targeting moiety that binds to a receptor that is expressed in tumor neovasculature, an optional linking group, and a radioactive metal ion that emits ionizing radiation such as beta particles, alpha particles and Auger or Coster-Kronig electrons. The receptor binding compounds target the radioisotope to the tumor neovasculature. The beta or alpha-particle emitting radioisotope emits a cytotoxic amount of ionizing radiation which results in cell death. The penetrating ability of radiation obviates the requirement that the cytotoxic agent diffuse or be transported into the cell to be cytotoxic.
It is another object of the present invention to provide pharmaceuticals to treat rheumatoid arthritis. These pharmaceuticals comprise a targeting moiety that gbinds to a receptor that is upregulated during angiogenesis, an optional linking group, and a radioisotope athat emits cytotoxic radiation (i.e., beta particles, alpha particles and Auger or Coster-Kronig electrons). In rheumatoid arthritis, the ingrowth of a highly vascularized pannus is caused by the excessive production of angiogenic factors by the infiltrating macrophages, immune cells, or inflammatory cells. Therefore, the radiopharmaceuticals of the present invention that emit cytotoxic radiation could be used to destroy the new angiogenic vasculature that results and thus treat the disease.
It is another object of the present invention to provide tumor imaging agents, comprised of targeting moiety that binds to a receptor that is upregulated during angiogenesis, an optional linking group, and an imageable moiety, such as a gamma ray or positron emitting radioisotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.
It is another object of the present invention to provide imaging agents for monitoring the progress and results of therapeutic angiogenesis treatment. These agents comprise of targeting moiety that binds to a receptor that is upregulated during angiogenesis, an optional linking group, and an imageable moiety. Imaging agents of the present invention could be administered intravenously periodically after the administration of growth factors and imaging would be performed using standard techniques of the affected areas, heart or limbs, to monitor the progress and results of the therapeutic angiogenesis treatment (i.e., image the formation of new blood vessels).
It is another object of the present invention to provide compounds useful for preparing the pharmaceuticals of the present invention. These compounds are comprised of a peptide or peptidomimetic targeting moiety that binds to a receptor that is upregulated during angiogenesis, Q, an optional linking group, Ln, and a metal chelator or bonding moiety, Ch. The compounds may have one or more protecting groups attached to the metal chelator or bonding moiety. The protecting groups provide improved stability to the reagents for long-term storage and are removed either iimmediately prior to or concurrent with the synthesis of the radiopharmaceuticals. Alternatively, the compounds of the present invention are comprised of a peptide or peptidomimetic targeting moiety that binds to a receptor that is upregulated during angiogenesis, Q, an optional linking group, Ln, and a surfactant, Sf.
The pharmaceuticals of the present invention may be used for diagnostic and/or therapeutic purposes. Diagnostic radiopharmaceuticals of the present invention are pharmaceuticals comprised of a diagnostically useful radionuclide (i.e., a radioactive metal ion that has imageable gamma ray or positron emissions). Therapeutic radiopharmaceuticals of the present invention are pharmaceuticals comprised of a therapeutically useful radionuclide, a radioactive metal ion that emits ionizing radiation such as beta particles, alpha particles and Auger or Coster-Kronig electrons.
The pharmaceuticals comprising a gamma ray or positron emitting radioactive metal ion are useful for imaging tumors by gamma scintigraphy or positron emission tomography. The pharmaceuticals comprising a gamma ray or positron emitting radioactive metal ion are also useful for imaging therapeutic angiogenesis by gamma scintigraphy or positron emission tomography. The pharmaceuticals comprising a particle emitting radioactive metal ion are useful for treating cancer by delivering a cytotoxic dose of radiation to the tumors. The pharmaceuticals comprising a particle emitting radioactive metal ion are also useful for treating rheumatoid arthritis by destroying the formation of angiogenic vasculature. The pharmaceuticals comprising a paramagnetic metal ion are useful as magnetic resonance imaging contrast agents. The pharmaceuticals comprising one or more X-ray absorbing or xe2x80x9cheavyxe2x80x9d atoms of atomic number 20 or greater are useful as X-ray contrast agents. The pharmaceuticals comprising a microbubble of a biocompatible gas, a liquid carrier, and a surfactant microsphere, are useful as ultrasound contrast agents.
[1] Thus, in a first embodiment, the present invention provides a novel compound, comprising: a targeting moiety and a chelator, wherein the targeting moiety is bound to the chelator, is a benzodiazepine nonpeptide, and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and chelator.
[2] In a preferred embodiment, the targeting moiety comprises a benzodiazepine and the receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, xcex1vxcex23, xcex1vxcex25, xcex15xcex21, xcex14xcex21, xcex11xcex21, and xcex12xcex22 and the linking group is present between the targeting moiety and chelator.
[3] In a more preferred embodiment, the receptor is the integrin xcex1vxcex23 and the compound is of the formula:
(Q)dxe2x80x94Lnxe2x80x94Ch or (Q)dxe2x80x94Lnxe2x80x94(Ch)dxe2x80x2
wherein, Q is a compound of Formula (I): 
wherein:
one of R or R1 is selected f rom a bond to Ln or (CH2)1-4 or an NH bond to Ln and the other of R or R1 is selected from C1-4 alkyl, benzyl or phenethyl;
R2 is selected from benzimidazole or imidazole;
R3 is selected from H, C1-4 alkyl or benzyl;
R4 is selected from H, C1-4 alkyl or benzyl;
d is selected from 1, 2 and 3;
Ln is a linking group having the formula: 
provided that g+h+gxe2x80x2+k+hxe2x80x2+gxe2x80x3+hxe2x80x3+gxe2x80x2xe2x80x3 is other than 0;
W is independently selected at each occurrence from the group: O, S, NH, NHC(xe2x95x90O), C(xe2x95x90O)NH, C(xe2x95x90O), C(xe2x95x90O)O, OC(xe2x95x90O), NHC(xe2x95x90S)NH, NHC(xe2x95x90O)NH, SO2(OCH2CH2)s, (CH2CH2O)sxe2x80x2, (OCH2CH2CH2)s, (CH2CH2CH2O)t, and (aa)txe2x80x2;
aa is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3 R10, C3-10 cycloalkyl substituted with 0-3 R10, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R10;
R6, R6a, R7, R7a, R8, R8a, R8b, R9, R9a and R9b are independently selected at each occurrence from the group: H, xe2x95x90O, COOH, SO3H, PO3H, C1-C5 alkyl substituted with 0-3 R10, aryl substituted with 0-3 R10, benzyl substituted with 0-3 R10, and C1-C5 alkoxy substituted with 0-3 R10, NHC(xe2x95x90O)R11, C(xe2x95x90O)NHR11, NHC(xe2x95x90O) NHR11, NHR11, R11, and a bond to Ch;
R10 is independently selected at each occurrence from the group: a bond to Ch, COOR11, OH, NHR11, C(xe2x95x90O)NHR11, NH(Cxe2x95x90O)R11,SO3H, PO3H, xe2x95x90O, R11, aryl substituted with 0-3 R11, C1-5 alkyl substituted with 0-1 R12, C1-5 alkoxy substituted with 0-1 R12, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R11;
R11 is independently selected at each occurrence from the group: H, C1-C10 alkyl substituted with 0-1 R12, aryl substituted with 0-1 R12, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R12, C3-10 cycloalkyl substituted with 0-1 R12, polyalkylene glycol substituted with 0-1 R12, carbohydrate substituted with 0-1 R12, cyclodextrin substituted with 0-1 R12, amino acid substituted with 0-1 R12, polycarboxyalkyl substituted with 0-1 R12, polyazaalkyl substituted with 0-1 R12, peptide substituted with 0-1 R12, wherein the peptide is comprised of 2-10 amino acids, and a bond to Ch;
R12 is a bond to Ch;
k is selected from 0, 1, and 2;
h is selected from 0, 1, and 2;
hxe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
hxe2x80x3 is selected from 0, 1, 2, 3, 4, and 5;
hxe2x80x3xe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x3xe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x3xe2x80x3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
sxe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
sxe2x80x3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
txe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Ch is a metal bonding unit having a formula selected from the group: 
A1, A2, A3, A4, A5, A6, A7, and A8 are independently selected at each occurrence from the group: NR13, NR13R14, S, SH, S(Pg), O, OH, PR13, PR13R14, P(O)R15R16, CO2H and a bond to Ln;
E is a bond, CH, or a spacer group independently selected at each occurrence from the group: C1-C10 alkyl substituted with 0-3 R171, aryl substituted with 0-3 R17, C3-10 cycloalkyl substituted with 0-3 R17, heterocyclo-C1-10 alkyl substituted with 0-3 R17, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C6-10 aryl-C1-10 alkyl substituted with 0-3 R17, C110 alkyl-C6-10 aryl-substituted with 0-3 R17, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R17;
R13, and R14 are each independently selected from the group: a bond to Ln, hydrogen, C1-C10 alkyl substituted with 0-3 R17, aryl substituted with 0-3 R17, C1-10 cycloalkyl substituted with 0-3 R17, heterocyclo-C1-10 alkyl substituted with 0-3 R17, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C6-10 aryl-C1-10 alkyl substituted with 0-3 R17, C1-10 alkyl-C6-10 aryl-substituted with 0-3 R17, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R17, and an electron, provided that when one of R13 or R14 is an electron, then the other is also an electron;
alternatively, R13 and R14 combine to form xe2x95x90C(R20) (R21);
R15 and R16 are each independently selected from the group: a bond to Ln, xe2x80x94OH, C1-C10 alkyl substituted with 0-3 R17, C1-C10 alkyl substituted with 0-3 R17, aryl substituted with 0-3 R17, C3-10 cycloalkyl substituted with 0-3 R17, heterocyclo-C1-10 alkyl substituted with 0-3 R17, wherein the heterocyclo group is a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O, C6-10 aryl-C1-10 alkyl substituted with 0-3 R17, C1-10 alkyl-C6-10 aryl-substituted with 0-3 R17, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R17;
R17 is independently selected at each occurrence from the group: a bond to Ln, xe2x95x90O, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94CN, xe2x80x94CO2R18, xe2x80x94C(xe2x95x90O)R18, xe2x80x94C(xe2x95x90O)N(R18)2, xe2x80x94CHO, xe2x80x94CH2OR18, xe2x80x94OC(xe2x95x90O)R18, xe2x80x94OC(xe2x95x90O)OR18a, xe2x80x94OR18, xe2x80x94OC(xe2x95x90O)N(R18)2, xe2x80x94NR19C(xe2x95x90O)R18, xe2x80x94NR19C(xe2x95x90O)OR18a, xe2x80x94NR19C(xe2x95x90O)N(R18)2, xe2x80x94NR19SO2N(R18)2, xe2x80x94NR19SO2R18a, xe2x80x94SO3H, xe2x80x94SO2R18a, xe2x80x94SR18, xe2x80x94S(xe2x95x90O)R18a, xe2x80x94SO2N(R18)2, xe2x80x94N(R18)2, xe2x80x94NHC(xe2x95x90S)NHR18, xe2x95x90NOR18, NO2, xe2x80x94C(xe2x95x90O)NHOR18, xe2x80x94C(xe2x95x90O)NHNR18R18a, xe2x80x94OCH2CO2H, 2-(1-morpholino)ethoxy, C1-C5 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C2-C6 alkoxyalkyl, aryl substituted with 0-2 R18, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O;
R18, R18a, and R19 are independently selected at each occurrence from the group: a bond to Ln, H, C1-C6 alkyl, phenyl, benzyl, C1-C6 alkoxy, halide, nitro, cyano, and trifluoromethyl;
Pg is a thiol protecting group;
R20 and R21 are independently selected from the group: H, C1-C10 alkyl, xe2x80x94CN, xe2x80x94CO2R25, xe2x80x94C(xe2x95x90O)R25, xe2x80x94C(xe2x95x90O)N(R25)2, C2-C10 1-alkene substituted with 0-3 R23, C2-C10 1-alkyne substituted with 0-3 R23, aryl substituted with 0-3 R23, unsaturated 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R23, and unsaturated C3-10 carbocycle substituted with 0-3 R23;
alternatively, R20 and R21, taken together with the divalent carbon radical to which they are attached form: 
R22 and R23 are independently selected from the group: H, R24, C1-C10 alkyl substituted with 0-3 R24, C2-C10 alkenyl substituted with 0-3 R24, C2-C10 alkynyl substituted with 0-3 R24, aryl substituted with 0-3 R24, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R24, and C3-10 carbocycle substituted with 0-3 R24;
alternatively, R22, R23 taken together form a fused aromatic or a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O;
a and b indicate the positions of optional double bonds and n is 0 or 1;
R24 is independently selected at each occurrence from the group: xe2x95x90O, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94CN, xe2x80x94CO2R25, xe2x80x94C(xe2x95x90O)R25, xe2x80x94C(xe2x95x90O)N(R25)2, xe2x80x94N(R25)3+, xe2x80x94CH2OR25, xe2x80x94OC(xe2x95x90O)R25, xe2x80x94OC(xe2x95x90O)OR25a, xe2x80x94OR25, xe2x80x94OC(xe2x95x90O)N(R25)2, xe2x80x94NR26C(xe2x95x90O)R25, xe2x80x94NR26C(xe2x95x90O)OR25a, xe2x80x94NR26C(xe2x95x90O)N(R25)2, xe2x80x94NR26SO2N(R25)2, xe2x80x94NR26SO2R25a, xe2x80x94SO3H, xe2x80x94SO2R25a, xe2x80x94SR25, xe2x80x94S(xe2x95x90O)R25a, xe2x80x94SO2N(R25)2, xe2x80x94N(R25)2, xe2x95x90NOR25, xe2x80x94C(xe2x95x90O)NHOR25, xe2x80x94OCH2CO2H, and 2-(1-morpholino)ethoxy; and,
R25, R25a, and R26 are each independently selected at each occurrence from the group: hydrogen and C1-C6 alkyl;
and a pharmaceutically acceptable salt thereof.
[4] In an even more preferred embodiment, the present invention provides a compound wherein:
d is selected from 1, 2, 3, 4, and 5;
Z is selected from the group: aryl substituted with 0-1 R10, C3-10 cycloalkyl substituted with 0-1 R10, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R10;
A1, A2, A3, A4, A5, A6, A7, and A8 are independently selected at each occurrence from the group: NR13, NR13R14, S, SH, S(Pg), OH, and a bond to Ln;
E is a bond, CH, or a spacer group independently selected at each occurrence from the group: C1-C10 alkyl substituted with 0-3 R17, aryl substituted with 0-3 R17, C3-10 cycloalkyl substituted with 0-3 R17, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N. S, and O and substituted with 0-3 R17;
R13, and R14 are each independently selected from the group: a bond to Ln, hydrogen, C1-C10 alkyl substituted with 0-3 R17, aryl substituted with 0-3 R17, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R17, and an electron, provided that when one of R13 or R14 is an electron, then the other is also an electron;
alternatively, R13 and R14 combine to form xe2x95x90C(R20) (R21);
R17 is independently selected at each occurrence from the group: a bond to Ln, xe2x95x90O, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94CN, xe2x80x94CO2R18, xe2x80x94C(xe2x95x90O)R18, xe2x80x94C(xe2x95x90O)N(R18)2, xe2x80x94CH2OR18, xe2x80x94OC(xe2x95x90O)R18, xe2x80x94OC(xe2x95x90O)OR18a, xe2x80x94OR18, xe2x80x94OC(xe2x95x90O)N(R18)2, xe2x80x94NR19C(xe2x95x90O)R18, xe2x80x94NR19C(xe2x95x90O)OR18a, xe2x80x94NR19C(xe2x95x90O)N(R18)2, xe2x80x94NR19SO2N(R18)2, xe2x80x94NR19SO2R18a, xe2x80x94SO3H, xe2x80x94SO2R18a, xe2x80x94S(xe2x95x90O)R18a, xe2x80x94SO2N(R18)2, xe2x80x94N(R18)2, xe2x80x94NHC(xe2x95x90S)NHR18, xe2x95x90NOR18, xe2x80x94C(xe2x95x90O)NHNR18R18a, xe2x80x94OCH2CO2H, and 2-(1-morpholino)ethoxy;
R18, R18a, and R19 are independently selected at each occurrence from the group: a bond to Ln, H, and C1-C6 alkyl;
R20 and R21 are independently selected from the group: H, C1-C5 alkyl, xe2x80x94CO2R25, C2-C5 1-alkene substituted with 0-3 R23, C2-C5 1-alkyne substituted with 0-3 R23, aryl substituted with 0-3 R23, and unsaturated 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R23;
alternatively, R20 and R21, taken together with the divalent carbon radical to which they are attached form: 
R22 and R23 are independently selected from the group: H, and R24;
alternatively, R22, R23 taken together form a fused aromatic or a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O;
R24 is independently selected at each occurrence from the group: xe2x80x94CO2R25, xe2x80x94C(xe2x95x90O)N(R25)2, xe2x80x94CH2OR25, xe2x80x94OC(xe2x95x90O)R25, xe2x80x94OR25, xe2x80x94SO3H, xe2x80x94N(R25)2, and xe2x80x94OCH2CO2H; and,
R25 is independently selected at each occurrence from the group: H and C1-C3 alkyl.
[5] In a still more preferred embodiment, the present invention provides a compound, wherein:
Q is a peptide selected from the group: 
K is an L-amino acid independently selected at each occurrence from the group: arginine, citrulline, N-methylarginine, lysine, homolysine, 2-aminoethylcysteine, d-N-2-imidazolinylornithine, d-N-benzylcarbamoylornithine, and b-2-benzimidazolylacetyl-1,2-diaminopropionic acid;
L is glycine;
M is L-aspartic acid;
Mxe2x80x2 is D-aspartic acid;
R1 is L-valine, D-valine or L-lysine optionally substituted on the e amino group with a bond to Ln;
R2 is L-phenylalanine, D-phenylalanine, D-1-naphthylalanine, 2-aminothiazole-4-acetic acid or tyrosine, the tyrosine optionally substituted on the hydroxy group with a bond to Ln;
R3 is D-valine;
R4 is D-tyrosine substituted on the hydroxy group with a bond to Ln;
provided that one of R1 and R2 in each Q is substituted with a bond to Ln, and further provided that when R2 is 2-aminothiazole-4-acetic acid, K is N-methylarginine;
provided that at least one Q is a benzodiazepine;
d is 1, 2, or 3;
A1 is selected from the group: OH, and a bond to Ln;
A2, A4, and A6 are each N;
A3, A5, and A8 are each OH;
A7 is a bond to Ln or NH-bond to Ln;
E is a C2 alkyl substituted with 0-1 R17;
R17 is xe2x95x90O;
alternatively, Ch is 
is selected from the group: OH, and a bond to Ln;
A1, A2, A3 and A4 are each N;
A5, A6 and A8 are each OH;
A7 is a bond to Ln;
E is a C2 alkyl substituted with 0-1 R17;
R17 is xe2x95x90O;
alternatively, Ch is 
A1 is NH2 or Nxe2x95x90C(R20)(R21);
E is a bond;
A2 is NHR13;
R13 is a heterocycle substituted with R17, the heterocycle being selected from pyridine and pyrimidine;
R17 is selected from a bond to Ln, C(xe2x95x90O)NHR18 and C(xe2x95x90O)R18;
R18 is a bond to Ln;
R24 is selected from the group: xe2x80x94CO2R25, xe2x80x94OR25, xe2x80x94SO3H, and xe2x80x94N(R25)2; and,
R25 is independently selected at each occurrence from the group: hydrogen and methyl.
[6] In another even more preferred embodiment, the present invention provides a compound selected from the group:
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid;
(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid;
(S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl) benzenesulfonic acid;
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino)butanoic acid;
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid;
(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl) carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino) butanoic acid;
(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl carbamoyl)-5-((methoxycarbonyl)imethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)butanoic acid;
(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino) propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl) propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxy methyl)cyclododecyl)acetylamino) hexanoylamino)butanoic acid;
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy hexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl) acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo {Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl) carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoic acid; and
or a pharmaceutically acceptable salt form thereof.
[7] In a further preferred embodiment, the present invention provides a kit comprising a compound of the present invention.
[8] In an even further preferred embodiment, the kit further comprises one or more ancillary ligands and a reducing agent.
[9] In a still further preferred embodiment, the ancillary ligands are tricine and TPPTS.
[10] In another still further preferred embodiment, the reducing agent is tin(II).
[11] In a second embodiment, the present invention provides a novel diagnostic or therapeutic metallopharmaceutical composition, comprising: a metal, a chelator capable of chelating the metal and a targeting moiety, wherein the targeting moiety is bound to the chelator, is a benzodiazepine nonpeptide and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and chelator.
[12] In another preferred embodiment, the metallopharmaceutical is a diagnostic radiopharmaceutical, the metal is a radioisotope selected from the group: 99mTc, 95Tc, 111In, 62Cu, 64Cu, 67Ga, and 68Ga, the targeting moiety comprises a benzodiazepine and the receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, xcex1vxcex23, xcex1vxcex25, xcex15xcex21, xcex14xcex21, xcex11xcex21, and xcex12xcex22 and the linking group is present between the targeting moiety and chelator.
[13] In another more preferred embodiment, the targeting moiety is a benzodiazepine and the receptor is xcex1vxcex23.
[14] In another even more preferred embodiment, the radioisotope is 99mTc or 95Tc, the radiopharmaceutical further comprises a first ancillary ligand and a second ancillary ligand capable of stabilizing the radiopharmaceutical.
[15] In another still more preferred embodiment, the radioisotope is 99mTc.
[16] In another further preferred embodiment, the radiopharmaceutical is selected from the group:
99mTc((S)-2-(2,5-diaza-5-(6((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid)(tricine)(TPPTS);
99mTc((S)-2-(2,5-diaza-9-(N-(6-((6-(diazenido)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(tricine)(TPPTS);
[17] In another even more preferred embodiment, the radioisotope is 111In.

[18] In another still more preferred embodiment, the radiopharmaceutical is selected from the group:
111In complex of 6-(N-(3-(3-aza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-((carboxymethyl)(2-((carboxymethyl)methylamino)ethyl)amino) ethyl)(2-((carboxymethyl)ethylamino)ethyl)amino)-acetylamino)-4-oxooctane-1,8-dicarboxylic acid;
11In complex of (S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid; and
111In complex of (S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl) cyclododecyl)acetylamino)propanoic acid.
[19] In another preferred embodiment, the metallopharmaceutical is a therapeutic radiopharmaceutical, the metal is a radioisotope selected from the group: 186Re, 188Re, 153Sm, 166Ho, 177Lu, 149Pm, 90Y, 212Bi, 103Pd, 109Pd, 159Gd, 140La, 198Au, 199Au, 169Yb, 175Yb, 165Dy, 166Dy, 67Cu, 105Rh, 111Ag, and 192Ir, the targeting moiety is a benzodiazepine nonpeptide and the receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, xcex1vxcex23, xcex1vxcex25, xcex15xcex21, xcex14xcex21, xcex11xcex21, and xcex12xcex22 and the linking group is present between the targeting moiety and chelator.
[20] In another more preferred embodiment, the targeting moiety is an benzodiazepine and the receptor is xcex1vxcex23.
[21] In another even more preferred embodiment, the radioisotope is 153Sm.
[22] In another even more preferred embodiment, the radioisotope is 177Lu.
[23] In another even more preferred embodiment, the radioisotope is 90Y.
[24] In another preferred embodiment, the metallopharmaceutical is a MRI contrast agent, the metal is a paramagnetic metal ion selected from the group: Gd(III), Dy(III), Fe(III), and Mn(II), the targeting moiety is a benzodiazepine nonpeptide and the receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, xcex1vxcex23, xcex1vxcex25, xcex15xcex21, xcex14xcex21, xcex11xcex21, and xcex12xcex22 and the linking group is present between the targeting moiety and chelator.
[25] In another more preferred embodiment, the targeting moiety is a benzodiazepine and the receptor is xcex1vxcex23.
[26] In another even more preferred embodiment, the metal ion is Gd(III).
[27] In another preferred embodiment, the metallopharmaceutical is a X-ray contrast agent, the metal is selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pdf Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir, the targeting moiety is a benzodiazepine nonpeptide, the receptor is xcex1vxcex23, and the linking group is present between the targeting moiety and chelator.
[28] In another even more preferred embodiment, the present invention provides a novel method of treating rheumatoid arthritis in a patient comprising: administering a therapeutic radiopharmaceutical of the present invention capable of localizing in new angiogenic vasculature to a patient by injection or infusion.
[29] In another even more preferred embodiment, the present invention provides a novel method of treating cancer in a patient comprising: administering to a patient in need thereof a therapeutic radiopharmaceutical of the present invention by injection or infusion.
[30] In another even more preferred embodiment, the present invention provides a novel method of imaging formation of new blood vessels in a patient comprising: (1) administering a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of the present invention to a patient by injection or infusion; (2) imaging the area of the patient wherein the desired formation of new blood vessels is located.
[31] In another even more preferred embodiment, the present invention provides a novel method of imaging cancer in a patient comprising: (1) administering a diagnostic radiopharmaceutical of the present invention to a patient by injection or infusion; (2) imaging the patient using planar or SPECT gamma scintigraphy, or positron emission tomography.
[32] In another even more preferred embodiment, the present invention provides a novel method of imaging cancer in a patient comprising: (1) administering a MRI contrast agent of the present invention; and (2) imaging the patient using magnetic resonance imaging.
[33] In another even more preferred embodiment, the present invention provides a novel method of imaging cancer in a patient comprising: (1) administering a X-ray contrast agent of the present invention; and (2) imaging the patient using X-ray computed tomography.
[34] In a third embodiment, the present invention provides a novel compound capable of being used in an ultrasound contrast composition, comprising: a targeting moiety and a surfactant, wherein the targeting moiety is bound to the surfactant, is a benzodiazepine nonpeptide, and binds to a receptor that is upregulated during angiogenesis and the compound has 0-1 linking groups between the targeting moiety and surfactant.
[35] In a preferred embodiment, the targeting moiety comprises an benzodiazepine and the receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl, xcex1vxcex23, xcex1vxcex25, xcex15xcex21, xcex14xcex21, xcex11xcex21, and xcex12xcex22 and the linking group is present between the targeting moiety and surfactant.
[36] In a more preferred embodiment, the receptor is the integrin xcex1vxcex23 and the compound is of the formula:
(Q)dxe2x80x94Lnxe2x80x94Sf
wherein, Q is a compound of Formula (I): 
wherein:
one of R or R1 is selected from a bond to Ln or (CH2)1-4 or an NH bond to Ln and the other of R or R1 is selected from C1-4 alkyl, benzyl or phenethyl;
R2 is selected from benzimidazole or imidazole;
R3 is selected from H, C1-4 alkyl or benzyl;
R4 is selected from H, C1-4 alkyl or benzyl;
d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
Sf is a surfactant which is a lipid or a compound of the formula 
A9 is selected from the group: OH and OR27;
A10 is OR27;
R27 is C(xe2x95x90O)C1-20 alkyl;
E1 is C1-10 alkylene substituted with 1-3 R28;
R28 is independently selected at each occurrence from the group: R30, xe2x80x94PO3Hxe2x80x94R30, xe2x95x90O, xe2x80x94CO2R29, xe2x80x94C(xe2x95x90O)R29, xe2x80x94C(xe2x95x90O)N(R29)2, xe2x80x94CH2OR29, xe2x80x94OR29, xe2x80x94N(R29)2, C1-C5 alkyl, and C2-C4 alkenyl;
R29 is independently selected at each occurrence from the group: R30, H, C1-C6 alkyl, phenyl, benzyl, and trifluoromethyl;
R30 is a bond to Ln;
Ln is a linking group having the formula:
(CR6R7)gxe2x80x94(W)hxe2x80x94(CR6aR7a)gxe2x80x2xe2x80x94(Z)kxe2x80x94(W)hxe2x80x2xe2x80x94(CR8R9)gxe2x80x3xe2x80x94(W)hxe2x80x3xe2x80x94(CR8aR9a)gxe2x80x3xe2x80x2
W is independently selected at each occurrence from the group: O, S, NH, NHC(xe2x95x90O), C(xe2x95x90O)NH, C(xe2x95x90O), C(xe2x95x90O)O, OC(xe2x95x90O), NHC(xe2x95x90S)NH, NHC(xe2x95x90O)NH, SO2, (OCH2CH2)20-200, (CH2CH2O)20-200, (OCH2CH2CH2)20-200, (CH2CH2CH2O)20-200, and (aa)txe2x80x2;
aa is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3 R10, C3-10 cycloalkyl substituted with 0-3 R10, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N. S, and O and substituted with 0-3 R10;
R6, R6a, R7, R7a, R8, R8a, R9 and R9a are independently selected at each occurrence from the group: H, xe2x95x90O, COOH, SO3H, PO3H, C1-C5 alkyl substituted with 0-3 R10, aryl substituted with 0-3 R10, benzyl substituted with 0-3 R10, and C1-C5 alkoxy substituted with 0-3 R10, NHC(xe2x95x90O)R11, C(xe2x95x90O)NHR11, NHC(xe2x95x90O)NHR11, NHR11, R11, and a bond to Sf;
R10 is independently selected at each occurrence from the group: a bond to Sf, COOR11, OH, NHR11, SO3H, PO3H, aryl substituted with 0-3 R11, C0-5 alkyl substituted with 0-1 R12, C1-5 alkoxy substituted with 0-1 R12, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R11;
R11 is independently selected at each occurrence from the group: H, aryl substituted with 0-1 R12, a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-1 R12, C3-10 cycloalkyl substituted with 0-1 R12, amino acid substituted with 0-1 R12, and a bond to Sf;
R12 is a bond to Sf;
k is selected from 0, 1, and 2;
h is selected from 0, 1, and 2;
hxe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
hxe2x80x3 is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x3 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
gxe2x80x3xe2x80x2is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10,
txe2x80x2 is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
and a pharmaceutically acceptable salt thereof.
[37] In another even more preferred embodiment, the compound is of the formula:
Qxe2x80x94Lnxe2x80x94Sf
wherein, Q is a compound of Formula (I): 
wherein:
one of R or R1 is selected from a bond to Ln or (CH2)1-4 or an NH bond to Ln and the other of R or R1 is selected from C1-4 alkyl, benzyl or phenethyl;
R2 is selected from benzimidazole or imidazole;
R3 is selected from H, C1-4 alkyl or benzyl;
R4 is selected from H, C1-4 alkyl or benzyl;
Sf is a surfactant which is a lipid or a compound of the formula: 
A9 is OR27;
A10 is OR27;
R27 is C(xe2x95x90O)C1-15 alkyl;
E1 is C1-4 alkylene substituted with 1-3 R28;
R28 is independently selected at each occurrence from the group: R30, xe2x80x94PO3Hxe2x80x94R30, xe2x95x90O, xe2x80x94CO2R29, xe2x80x94C(xe2x95x90O)R29, xe2x80x94CH2OR29, xe2x80x94OR29, and C1-C5 alkyl;
R29 is independently selected at each occurrence from the group: R30, H, C1-C6 alkyl, phenyl, and benzyl;
R30 is a bond to Ln;
Ln is a linking group having the formula: 
W is independently selected at each occurrence from the group: O, S, NH, NHC(xe2x95x90O), C(xe2x95x90O)NH, C(xe2x95x90O), C(xe2x95x90O)O, OC(xe2x95x90O), NHC(xe2x95x90S)NH, NHC(xe2x95x90O)NH, SO2, (OCH2CH2)20-200, (CH2CH2Q)20-200, (OCH2CH2CH2)20-200, (CH2CH2CH2O)20-200, and (aa)txe2x80x2;
aa is independently at each occurrence an amino acid;
Z is selected from the group: aryl substituted with 0-3 R10, C3-10 cycloalkyl substituted with 0-3 R10, and a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and O and substituted with 0-3 R10;
R6, R6a, R7, R7a, R8, R8a, R9 and R9a are independently selected at each occurrence from the group: H, xe2x95x90O, C1-C5 alkyl substituted with 0-3 R10, and C1-C5 alkoxy substituted with 0-3 R10, and a bond to Sf;
R10 is independently selected at each occurrence from the group; a bond to Sf, COOR11, OH, NHR11, C1-5 alkyl substituted with 0-1 R12, and C1-5 alkoxy substituted with 0-1 R12;
R11 is independently selected at each occurrence from the group: H, aryl substituted with 0-1 R12, C3-10 cycloalkyl substituted with 0-1 R12, amino acid substituted with 0-1 R12, and a bond to Sf;
R12 is a bond to Sf;
k is selected from 0, 1, and 2;
h is selected from 0, 1, and 2;
hxe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
hxe2x80x3 is selected from 0, 1, 2, 3, 4, and 5;
g is selected from 0, 1, 2, 3, 4, and 5;
gxe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
gxe2x80x3 is selected from 0, 1, 2, 3, 4, and 5;
gxe2x80x3xe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
s is selected from 0, 1, 2, 3, 4, and 5;
sxe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
sxe2x80x3 is selected from 0, 1, 2, 3, 4, and 5;
t is selected from 0, 1, 2, 3, 4, and 5;
txe2x80x2 is selected from 0, 1, 2, 3, 4, and 5;
and a pharmaceutically acceptable salt thereof.
[38] In another still more preferred embodiment, the present invention provides a compound selected from the group:
Sodium 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid-dodecoanoate conjugate;
DPPE-PEG3400-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid]-dodecoanoate conjugate; and
[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)carbamoyl)propyl)carbamoyl]-w-amino-PEG3400-dodecanoate-DPPE conjugate;
[39] In another even more preferred embodiment, the present invention provides a novel ultrasound contrast agent composition, comprising:
(a) a compound comprising: a benzodiazepine that binds to the integrin xcex1vxcex23, surfactant and a linking group between the benzodiazepine and the surfactant;
(b) a parenterally acceptable carrier; and,
(c) an echogenic gas.
[40] In another still more preferred embodiment, the ultrasound contrast agent further comprises: 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.
[41] In another further preferred embodiment, the echogenic gas is a C2-5 perf luorocarbon.
[42] In another even more preferred embodiment, the present invention provides a method of imaging cancer in a patient comprising: (1) administering, by injection or infusion, a ultrasound contrast agent composition of the present invention to a patient; and (2) imaging the patient using sonography.
[43] In another even more preferred embodiment, the present invention provides a novel method of imaging formation of new blood vessels in a patient comprising: (1) administering, by injection or infusion, a ultrasound contrast agent composition of the present invention to a patient; (2) imaging the area of the patient wherein the desired formation of new blood vessels is located.
[44] In another even more preferred embodiment, the present invention provides a novel therapeutic radiopharmaceutical composition, comprising:
(a) a therapeutic radiopharmaceutical of the present invention; and,
(b) a parenterally acceptable carrier.
[45] In another even more preferred embodiment, the present invention provides a novel therapeutic radiopharmaceutical composition, comprising:
(a) a diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray contrast agent of the present invention; and,
(b) a parenterally acceptable carrier.
Another aspect of the present invention are diagnostic kits for the preparation of radiopharmaceuticals useful as imaging agents for cancer. Diagnostic kits of the present invention comprise one or more vials containing the sterile, non-pyrogenic, formulation comprised of a predetermined amount of a reagent of the present invention, and optionally other components such as one or two ancillary ligands, reducing agents, transfer ligands, buffers, lyophilization aids, stabilization aids, solubilization aids and bacteriostats. The inclusion of one or more optional components in the formulation will frequently improve the ease of synthesis of the radiopharmaceutical by the practicing end user, the ease of manufacturing the kit, the shelf-life of the kit, or the stability and shelf-life of the radiopharmaceutical. The inclusion of one or two ancillary ligands is required for diagnostic kits comprising reagent comprising a hydrazine or hydrazone bonding moiety. The one or more vials that contain all or part of the formulation can independently be in the form of a sterile solution or a lyophilized solid.
Another aspect of the present invention contemplates a method of imaging cancer in a patient involving: (1) synthesizing a diagnostic radiopharmaceutical of the present invention, using a reagent of the present invention, capable of localizing in tumors; (2) administering said radiopharmaceutical to a patient by injection or infusion; (3) imaging the patient using planar or SPECT gamma scintigraphy, or positron emission tomography.
Another aspect of the present invention contemplates a method of imaging cancer in a patient involving: (1) administering a paramagnetic metallopharmaceutical of the present invention capable of localizing in tumors to a patient by injection or infusion; and (2) imaging the patient using magnetic resonance imaging.
Another aspect of the present invention contemplates a method of imaging cancer in a patient involving: (1) administering a X-ray contrast agent of the present invention capable of localizing in tumors to a patient by injection or infusion; and (2) imaging the patient using X-ray computed tomography.
Another aspect of the present invention contemplates a method of imaging cancer in a patient involving: (1) administering a ultrasound contrast agent of the present invention capable of localizing in tumors to a patient by injection or infusion; and (2) imaging the patient using sonography.
Another aspect of the present invention contemplates a method of treating cancer in a patient involving: (1) administering a therapeutic radiopharmaceutical of the present invention capable of localizing in tumors to a patient by injection or infusion.
The compounds herein described may have asymmetric centers. Unless otherwise indicated, all chiral, diastereomeric and racemic forms are included in the present invention. Many geometric isomers of olefins, Cxe2x95x90N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. It will be appreciated that compounds of the present invention contain asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Two distinct isomers (cis and trans) of the peptide bond are known to occur; both can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. The D and L-isomers of a particular amino acid are designated herein using the conventional 3-letter abbreviation of the amino acid, as indicated by the following examples: D-Leu, or L-Leu.
When any variable occurs more than one time in any substituent or in any formula, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R52, then said group may optionally be substituted with up to two R52, and R52 at each occurrence is selected independently from the defined list of possible R52. Also, by way of example, for the group xe2x80x94N(R53)2, each of the two R53 substituents on N is independently selected from the defined list of possible R53. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. When a bond to a substituent is shown to cross the bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring.
The term xe2x80x9cnonpeptidenxe2x80x9d means preferably less than three amide bonds in the backbone core of the targeting moiety or preferably less than three amino acids or amino acid mimetics in the targeting moiety.
The term xe2x80x9cmetallopharmaceuticalxe2x80x9d means a pharmaceutical comprising a metal. The metal is the cause of the imageable signal in diagnostic applications and the source of the cytotoxic radiation in radiotherapeutic applications. Radiopharmaceuticals are metallopharmaceuticals in which the metal is a radioisotope.
By xe2x80x9creagentxe2x80x9d is meant a compound of this invention capable of direct transformation into a metallopharmaceutical of this invention. Reagents may be utilized directly for the preparation of the metallopharmaceuticals of this invention or may be a component in a kit of this invention.
The term xe2x80x9cbinding agentxe2x80x9d means a metallopharmaceutical of this invention having affinity for and capable of binding to the vitronectin receptor. The binding agents of this invention have Ki less than 1000 nM.
By xe2x80x9cstable compoundxe2x80x9d or xe2x80x9cstable structurexe2x80x9d is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious pharmaceutical agent.
The term xe2x80x9csubstitutedxe2x80x9d, as used herein, means that one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom""s or group""s normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is keto (i.e., xe2x95x90O), then 2 hydrogens on the atom are replaced.
The term xe2x80x9cbondxe2x80x9d, as used herein, means either a single or double bond.
The term xe2x80x9csaltxe2x80x9d, as used herein, is used as defined in the CRC Handbook of Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla., 1984, as any substance which yields ions, other than hydrogen or hydroxyl ions. As used herein, xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refer to derivatives of the disclosed compounds modified by making acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
The phrase xe2x80x9cpharmaceutically acceptablexe2x80x9d is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington""s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
As used herein, xe2x80x9calkylxe2x80x9d is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, examples of which include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl; xe2x80x9ccycloalkylxe2x80x9d or xe2x80x9ccarbocyclexe2x80x9d is intended to include saturated and partially unsaturated ring groups, including mono-, bi- or poly-cyclic ring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and adamantyl; xe2x80x9cbicycloalkylxe2x80x9d or xe2x80x9cbicyclicxe2x80x9d is intended to include saturated bicyclic ring groups such as [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, and so forth.
As used herein, the term xe2x80x9calkenexe2x80x9d or xe2x80x9calkenylxe2x80x9d is intended to include hydrocarbon chains having the specified number of carbon atoms of either a straight or branched configuration and one or more unsaturated carbonxe2x80x94carbon bonds which may occur in any stable point along the chain, such as ethenyl, propenyl, and the like.
As used herein, the term xe2x80x9calkynexe2x80x9d or xe2x80x9calkynylxe2x80x9d is intended to include hydrocarbon chains having the specified number of carbon atoms of either a straight or branched configuration and one or more unsaturated carbon-carbon triple bonds which may occur in any stable point along the chain, such as propargyl, and the like.
As used herein, xe2x80x9carylxe2x80x9d or xe2x80x9caromatic residuexe2x80x9d is intended to mean phenyl or naphthyl, which when substituted, the substitution can be at any position.
As used herein, the term xe2x80x9cheterocyclexe2x80x9d or xe2x80x9cheterocyclic systemxe2x80x9d is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic ring which is saturated partially unsaturated or unsaturated (aromatic), and which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure. The heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. If specifically noted, a nitrogen in the heterocycle may optionally be quaternized. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1. As used herein, the term xe2x80x9caromatic heterocyclic systemxe2x80x9d is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic aromatic ring which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S. It is preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1.
Examples of heterocycles include, but are not limited to, 1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl, xcex2-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl., oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are not limited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl. Also included are fused ring and spiro compounds containing, for example, the above heterocycles.
As used herein, the term Malkarylin means an aryl group bearing an alkyl group of 1-10 carbon atoms; the term xe2x80x9caralkylxe2x80x9d means an alkyl group of 1-10 carbon atoms bearing an aryl group; the term xe2x80x9carylalkarylxe2x80x9d means an aryl group bearing an alkyl group of 1-10 carbon atoms bearing an aryl group; and the term xe2x80x9cheterocycloalkylxe2x80x9d means an alkyl group of 1-10 carbon atoms bearing a heterocycle.
A xe2x80x9cpolyalkylene glycolxe2x80x9d is a polyethylene glycol, polypropylene glycol or polybutylene glycol having a molecular weight of less than about 5000, terminating in either a hydroxy or alkyl ether moiety.
A xe2x80x9ccarbohydratexe2x80x9d is a polyhydroxy aldehyde, ketone, alcohol or acid, or derivatives thereof, including polymers thereof having polymeric linkages of the acetal type.
A xe2x80x9ccyclodextrinxe2x80x9d is a cyclic oligosaccharide. Examples of cyclodextrins include, but are not limited to, xcex1-cyclodextrin, hydroxyethyl-xcex1-cyclodextrin, hydroxypropyl-xcex1-cyclodextrin, carboxymethyl-xcex2-cyclodextrin, hydroxypropyl-xcex2-cyclodextrin, carboxymethyl-xcex2-cyclodextrin, dihydroxypropyl-xcex2-cyclodextrin, hydroxyethyl-xcex2-cyclodextrin, 2,6 di-O-methyl-xcex2-cyclodextrin, sulfated-xcex2-cyclodextrin, xcex3-cyclodextrin, hydroxypropyl-xcex3-cyclodextrin, dihydroxypropyl-xcex3-cyclodextrin, hydroxyethyl-xcex3-cyclodextrin, and sulfated xcex3-cyclodextrin.
As used herein, the term xe2x80x9cpolycarboxyalkylxe2x80x9d means an alkyl group having between two and about 100 carbon atoms and a plurality of carboxyl substituents; and the term xe2x80x9cpolyazaalkylxe2x80x9d means a linear or branched alkyl group having between two and about 100 carbon atoms, interrupted by or substituted with a plurality of amine groups.
A xe2x80x9creducing agentxe2x80x9d is a compound that reacts with a radionuclide, which is typically obtained as a relatively unreactive, high oxidation state compound, to lower its oxidation state by transferring electron(s) to the radionuclide, thereby making it more reactive. Reducing agents useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to stannous chloride, stannous fluoride, formamidine sulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous or ferrous salts. Other reducing agents are described in Brodack et. al., PCT Application 94/22496, which is incorporated herein by reference.
A xe2x80x9ctransfer ligandxe2x80x9d is a ligand that forms an intermediate complex with a metal ion that is stable enough to prevent unwanted side-reactions but labile enough to be converted to a metallopharmaceutical. The formation of the intermediate complex is kinetically favored while the formation of the metallopharmaceutical is thermodynamically favored. Transfer ligands useful in the preparation of metallopharmaceuticals and in diagnostic kits useful for the preparation of diagnostic radiopharmaceuticals include but are not limited to gluconate, glucoheptonate, mannitol, glucarate, N,N,Nxe2x80x2,Nxe2x80x2-ethylenediaminetetraacetic acid, pyrophosphate and methylenediphosphonate. In general, transfer ligands are comprised of oxygen or nitrogen donor atoms.
The term xe2x80x9cdonor atomxe2x80x9d refers to the atom directly attached to a metal by a chemical bond.
xe2x80x9cAncillaryxe2x80x9d or xe2x80x9cco-ligandsxe2x80x9d are ligands that are incorporated into a radiopharmaceutical during its synthesis. They serve to complete the coordination sphere of the radionuclide together with the chelator or radionuclide bonding unit of the reagent. For radiopharmaceuticals comprised of a binary ligand system, the radionuclide coordination sphere is composed of one or more chelators or bonding units from one or more reagents and one or more ancillary or co-ligands, provided that there are a total of two types of ligands, chelators or bonding units. For example, a radiopharmaceutical comprised of one chelator or bonding unit from one reagent and two of the same ancillary or co-ligands and a radiopharmaceutical comprised of two chelators or bonding units from one or two reagents and one ancillary or co-ligand are both considered to be comprised of binary ligand systems. For radiopharmaceuticals comprised of a ternary ligand system, the radionuclide coordination sphere is composed of one or more chelators or bonding units from one or more reagents and one or more of two different types of ancillary or co-ligands, provided that there are a total of three types of ligands, chelators or bonding units. For example, a radiopharmaceutical comprised of one chelator or bonding unit from one reagent and two different ancillary or co-ligands is considered to be comprised of a ternary ligand system.
Ancillary or co-ligands useful in the preparation of radiopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals are comprised of one or more oxygen, nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and tellurium donor atoms. A ligand can be a transfer ligand in the synthesis of a radiopharmaceutical and also serve as an ancillary or co-ligand in another radiopharmaceutical. Whether a ligand is termed a transfer or ancillary or co-ligand depends on whether the ligand remains in the radionuclide coordination sphere in the radiopharmaceutical, which is determined by the coordination chemistry of the radionuclide and the chelator or bonding unit of the reagent or reagents.
A xe2x80x9cchelatorxe2x80x9d or xe2x80x9cbonding unitxe2x80x9d is the moiety or group on a reagent that binds to a metal ion through the formation of chemical bonds with one or more donor atoms.
The term xe2x80x9cbinding sitexe2x80x9d means the site in vivo or in vitro that binds a biologically active molecule.
A xe2x80x9cdiagnostic kitxe2x80x9d or xe2x80x9ckitxe2x80x9d comprises a collection of components, termed the formulation, in one or more vials which are used by the practicing end user in a clinical or pharmacy setting to synthesize diagnostic radiopharmaceuticals. The kit provides all the requisite components to synthesize and use the diagnostic radiopharmaceutical except those that are commonly available to the practicing end user, such as water or saline for injection, a solution of the radionuclide, equipment for heating the kit during the synthesis of the radiopharmaceutical, if required, equipment necessary for administering the radiopharmaceutical to the patient such as syringes and shielding, and imaging equipment.
Therapeutic radiopharmaceuticals, X-ray contrast agent pharmaceuticals, ultrasound contrast agent pharmaceuticals and metallopharmaceuticals for magnetic resonance imaging contrast are provided to the end user in their final form in a formulation contained typically in one vial, as either a lyophilized solid or an aqueous solution. The end user reconstitutes the lyophilized with water or saline and withdraws the patient dose or just withdraws the dose from the aqueous solution formulation as provided.
A xe2x80x9clyophilization aidxe2x80x9d is a component that has favorable physical properties for lyophilization, such as the glass transition temperature, and is added to the formulation to improve the physical properties of the combination of all the components of the formulation for lyophilization.
A xe2x80x9cstabilization aidxe2x80x9d is a component that is added to the metallopharmaceutical or to the diagnostic kit either to stabilize the metallopharmaceutical or to prolong the shelf-life of the kit before it must be used. Stabilization aids can be antioxidants, reducing agents or radical scavengers and can provide improved stability by reacting preferentially with species that degrade other components or the metallopharmaceutical.
A xe2x80x9csolubilization aidxe2x80x9d is a component that improves the solubility of one or more other components in the medium required for the formulation.
A xe2x80x9cbacteriostatxe2x80x9d is a component that inhibits the growth of bacteria in a formulation either during its storage before use of after a diagnostic kit is used to synthesize a radiopharmaceutical. The following abbreviations are used herein:
The following conventional three-letter amino acid abbreviations are used herein; the conventional one-letter amino acid abbreviations are NOT used herein:
As used herein, the term xe2x80x9cbubblesxe2x80x9d, as used herein, refers to vesicles which are generally characterized by the presence of one or more membranes or walls surrounding an internal void that is filled with a gas or precursor thereto. Exemplary bubbles include, for example, liposomes, micelles and the like.
As used herein, the term xe2x80x9clipidxe2x80x9d refers to a synthetic or naturally-occurring amphipathic compound which comprises a hydrophilic component and a hydrophobic component. Lipids include, for example, fatty acids, neutral fats, phosphatides, glycolipids, aliphatic alchols and waxes, terpenes and steroids.
As used herein, the term xe2x80x9clipid compositionxe2x80x9d refers to a composition which comprises a lipid compound. Exemplary lipid compositions include suspensions, emulsions and vesicular compositions.
As used herein, the term xe2x80x9clipid formulationxe2x80x9d refers to a composition which comprises a lipid compound and a bioactive agent.
As used herein, the term xe2x80x9cvesiclexe2x80x9d refers to a spherical entity which is characterized by the presence of an internal void. Preferred vesicles are formulated from lipids, including the various lipids described herein. In any given vesicle, the lipids may be in the form of a monolayer or bilayer, and the mono- or bilayer lipids may be used to form one of more mono- or bilayers. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric. The lipid vesicles described herein include such entities commonly referred to as liposomes, micelles, bubbles, microbubbles, microspheres and the like. Thus, the lipids may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of about two or about three monolayers or bilayers) or a multilamellar vesicle (comprised of more than about three monolayers or bilayers). The internal void of the vesicles may be filled with a liquid, including, for example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid or solute material, including, for example, a bioactive agent, as desired.
As used herein, the term xe2x80x9cvesicular compositionxe2x80x9d refers to a composition which is formulate from lipids and which comprises vesicles.
As used herein, the term xe2x80x9cvesicle formulationsxe2x80x9d refers to a composition which comprises vesicles and a bioactive agent.
As used herein, the term xe2x80x9clipsomesxe2x80x9d refers to a generally spherical cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as lipid vesicles.
Angiogenesis is the process of formation of new capillary blood vessels from existing vasculature. It is an important component of a variety of physiological processes including ovulation, embryonic development, wound repair, and collateral vascular generation in the myocardium. It is also central to a number of pathological conditions such as tumor growth and metastasis, diabetic retinopathy, and macular degeneration. The process begins with the activation of existing vascular endothelial cells in response to a variety of cytokines and growth factors. The activated endothelial cells secrete enzymes that degrade the basement membrane of the vessels. The endothelial cells then proliferate and migrate into the extracellular matrix first forming tubules and subsequently new blood vessels.
Under normal conditions, endothelial cell proliferation is a very slow process, but it increases for a short period of time during embryogenesis, ovulation and wound healing. This temporary increase in cell turnover is governed by a combination of a number of growth stimulatory factors and growth suppressing factors. In pathological angiogenesis, this normal balance is disrupted resulting in continued increased endothelial cell proliferation. Some of the pro-angiogenic factors that have been identified include basic fibroblast growth factor (bFGF), angiogenin, TGF-alpha, TGF-beta, and vascular endothelium growth factor (VEGF), while interferon-alpha, interferon-beta and thrombospondin are examples of angiogenesis suppressors.
Angiogenic factors interact with endothelial cell surface receptors such as the receptor tyrosine kinases EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, and Axl. The receptors Flk-1/KDR, neuropilin-1, and Flt-1 recognize VEGF and these interactions play key roles in VEGF-induced angiogenesis. The Tie subfamily of receptor tyrosine kinases are also expressed prominently during blood vessel formation.
The proliferation and migration of endothelial cells in the extracellular matrix is mediated by interaction with a variety of cell adhesion molecules. Integrins are a diverse family of heterodimeric cell surface receptors by which endothelial cells attach to the extracellular matrix, each other and other cells. Angiogenesis induced by bFGF or TNF-alpha depend on the agency of the integrin avb3, while angiogenesis induced by VEGF depends on the integrin avb5 (Cheresh et. al., Science, 1995, 270, 1500-2). Induction of expression of the integrins a1b1 and a2b1 on the endothelial cell surface is another important mechanism by which VEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, Sci USA, 1997, 94, 13612-7).
The pharmaceuticals of the present invention are comprised of a non-peptide targeting moiety for the vitronectin receptor that is expressed or upregulated in angiogenic tumor vasculature.
The ultrasound contrast agents of the present invention comprise a plurality of vitronectin receptor targeting moieties attached to or incorporated into a microbubble of a biocompatible gas, a liquid carrier, and a surfactant microsphere, further comprising an optional linking moiety, Ln, between the targeting moieties and the microbubble. In this context, the term liquid carrier means aqueous solution and the term surfactant means any amphiphilic material which produces a reduction in interfacial tension in a solution. A list of suitable surfactants for forming surfactant microspheres is disclosed in EP0727225A2, herein incorporated by reference. The term surfactant microsphere includes nanospheres, liposomes, vesicles and the like. The biocompatible gas can be air, or a fluorocarbon, such as a C3-C5 perfluoroalkane, which provides the difference in echogenicity and thus the contrast in ultrasound imaging. The gas is encapsulated or contained in the microsphere to which is attached the biodirecting group, optionally via a linking group. The attachment can be covalent, ionic or by van der Waals forces. Specific examples of such contrast agents include lipid encapsulated perfluorocarbons with a plurality of tumor neovasculature receptor binding peptides, polypeptides or peptidomimetics.
X-ray contrast agents of the present invention are comprised of one or more vitronectin receptor targeting moieties attached to one or more X-ray absorbing or xe2x80x9cheavyxe2x80x9d atoms of atomic number 20 or greater, further comprising an optional linking moiety, Ln, between the targeting moieties and the X-ray absorbing atoms. The frequently used heavy atom in X-ray contrast agents is iodine. Recently, X-ray contrast agents comprised of metal chelates (Wallace, R., U.S. Pat. No. 5,417,959) and polychelates comprised of a plurality of metal ions (Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently, multinuclear cluster complexes have been disclosed as X-ray contrast agents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).
MRI contrast agents of the present invention are comprised of one or more vitronectin receptor targeting moieties attached to one or more paramagnetic metal ions, further comprising an optional linking moiety, Ln, between the targeting moieties and the paramagnetic metal ions. The paramagnetic metal ions are present in the form of metal complexes or metal oxide particles. U.S. Pat. No. 5,412,148, and 5,760,191, describe examples of chelators for paramagnetic metal ions for use in MRI contrast agents. U.S. Pat. No. 5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No. 5,281,704, describe examples of polychelants useful for complexing more than one paramagnetic metal ion for use in MRI contrast agents. U.S. Pat. No. 5,520,904, describes particulate compositions comprised of paramagnetic metal ions for use as MRI contrast agents.
The pharmaceuticals of the present invention have the formulae, (Q)dxe2x80x94Lnxe2x80x94(Chxe2x80x94X), (Q)dxe2x80x94Lnxe2x80x94(Chxe2x80x94X1)dxe2x80x2, (Q)dxe2x80x94Lnxe2x80x94(X2)dxe2x80x3, and (Q)dxe2x80x94Lnxe2x80x94(X3), wherein Q represents a non-peptide that binds to a receptor expressed in angiogenic tumor vasculature, d is 1-10, Ln represents an optional linking group, Ch represents a metal chelator or bonding moiety, X represents a radioisotope, X1 represents paramagnetic metal ion, X2 represents a paramagnetic metal ion or heavy atom containing insoluble solid particle, dxe2x80x2 is 1-100, and X3 represents a surfactant microsphere of an echogenic gas. The interaction of the non-peptide recognition sequences of the vitronectin receptor binding portion of the pharmaceuticals with the xcex1vxcex23 receptor results in localization of the pharmaceuticals in angiogenic tumor vasculature, which express the xcex1vxcex23 receptor.
The pharmaceuticals of the present invention can be synthesized by several approaches. One approach involves the synthesis of the targeting non-peptide moiety, Q, and direct attachment of one or more moieties, Q, to one or more metal chelators or bonding moieties, Ch, or to a paramagnetic metal ion or heavy atom containing solid particle, or to an echogenic gas microbubble. Another approach involves the attachment of one or more moieties, Q, to the linking group, Ln, which is then attached to one or more metal chelators or bonding moieties, Ch, or to a paramagnetic metal ion or heavy atom containing solid particle, or to an echogenic gas microbubble. Another approach involves the synthesis of a non-peptide, Q. bearing a fragment of the linking group, Ln, one or more of which are then attached to the remainder of the linking group and then to one or more metal chelators or bonding moieties, Ch, or to a paramagnetic metal ion or heavy atom containing solid particle, or to an echogenic gas microbubble.
The non-peptide vitronectin binding moieties, Q, optionally bearing a linking group, Ln, or a fragment of the linking group, can be synthesized using standard synthetic methods known to those skilled in the art. Preferred methods include but are not limited to those methods described below.
The attachment of linking groups, Ln, to the non-peptides, Q; chelators or bonding units, Ch, to the non-peptides, , Q, or to the linking groups, Ln; and non-peptides, bearing a fragment of the linking group to the remainder of the linking group, in combination forming the moiety, (Q)dxe2x80x94Ln, and then to the moiety Ch; can all be performed by standard techniques. These include, but are not limited to, amidation, esterification,.alkylation, and the formation of uireas or thioureas. Procedures for performing these attachments can be found in Brinkley, M., Bioconjugate Chemistry 1992, 3(1), which is incorporated herein by reference.
A number of methods can be used to attach the non-peptides, Q, to paramagnetic metal ion or heavy atom containing solid particles, X2, by one of skill in the art of the surface modification of solid particles. In general, the targeting moiety Q or the combination (Q)dLn is attached to a coupling group that react with a constituent of the surface of the solid particle. The coupling groups can be any of a number of silanes which react with surface hydroxyl groups on the solid particle surface, as described in co-pending PCT/U.S. 99/16804, and can also include polyphosphonates, polycarboxylates, polyphosphates or mixtures thereof which couple with the surface of the solid particles, as described in U.S. Pat. No. 5,520,904.
A number of reaction schemes can be used to attach the non-peptides, Q, to the surfactant microsphere, X3. These are illustrated in following reaction schemes where Sf represents a surfactant moiety that forms the surfactant microsphere.
Acylation Reaction:
Sfxe2x80x94C(xe2x95x90O)xe2x80x94Y+Qxe2x80x94NH2 or xe2x86x92Sfxe2x80x94C(xe2x95x90O)xe2x80x94NHxe2x80x94Q
Qxe2x80x94OH or Sfxe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94Q
Y is a leaving group or active ester
Disulfide Coupling:
Sfxe2x80x94SH+Qxe2x80x94SHxe2x86x92Sfxe2x80x94Sxe2x80x94Sxe2x80x94Q
Sulfonamide Coupling:
Sfxe2x80x94S(xe2x95x90O)2xe2x80x94Y+Qxe2x80x94NH2xe2x86x92Sfxe2x80x94S(xe2x95x90O)2xe2x80x94NHxe2x80x94Q
Reductive Amidation:
Sfxe2x80x94CHO+Qxe2x80x94NH2xe2x86x92Sfxe2x80x94NHxe2x80x94Q
In these reaction schemes, the substituents Sf and Q can be reversed as well.
The linking group Ln can serve several roles. First it provides a spacing group between the metal chelator or bonding moiety, Ch, the paramagnetic metal ion or heavy atom containing solid particle, X2, and the surfactant microsphere, X3, and the one or more of the non-peptides, Q, so as to minimize the possibility that the moieties Chxe2x80x94X, Chxe2x80x94X1, X2, and X3, will interfere with the interaction of the recognition sequences of Q with angiogenic tumor vasculature receptors. The necessity of incorporating a linking group in a reagent is dependent on the identity of Q, Chxe2x80x94X, Chxe2x80x94X1, X2, and X3. If Chxe2x80x94X, Chxe2x80x94X1, X2, and X3, cannot be attached to Q without substantially diminishing its affinity for the receptors, then a linking group is used. A linking group also provides a means of independently attaching multiple non-peptides, Q, to one group that is attached to Chxe2x80x94X, Chxe2x80x94X1, X2, or X3.
The linking group also provides a means of incorporating a pharmacokinetic modifier into the pharmaceuticals of the present invention. The pharmacokinetic modifier serves to direct the biodistibution of the injected pharmaceutical other than by the interaction of the targeting moieties, Q, with the vitronectin receptors expressed in the tumor neovasculature. A wide variety of functional groups can serve as pharmacokinetic modifiers, including, but not limited to, carbohydrates, polyalkylene glycols, peptides or other polyamino acids, and cyclodextrins. The modifiers can be used to enhance or decrease hydrophilicity and to enhance or decrease the rate of blood clearance. The modifiers can also be used to direct the route of elimination of the pharmaceuticals. Preferred pharmacokinetic modifiers are those that result in moderate to fast blood clearance and enhanced renal excretion.
The metal chelator or bonding moiety, Ch, is selected to form stable complexes with the metal ion chosen for the particular application. Chelators or bonding moieties for diagnostic radiopharmaceuticals are selected to form stable complexes with the radioisotopes that have imageable gamma ray or positron emissions, such as 99mTc, 95Tc, 111In, 62Cu, 6Cu, 64Cu, 67Ga, 68Ga, 86Y.
Chelators for technetium, copper and gallium isotopes are selected from diaminedithiols, monoamine-monoamidedithiols, triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, and hydrazines. The chelators are generally tetradentate with donor atoms selected from nitrogen, oxygen and sulfur. Preferred reagents are comprised of chelators having amine nitrogen and thiol sulfur donor atoms and hydrazine bonding units. The thiol sulfur atoms and the hydrazines may bear a protecting group which can be displaced either prior to using the reagent to synthesize a radiopharmaceutical or preferably in situ during the synthesis of the radiopharmaceutical.
Exemplary thiol protecting groups include those listed in Greene and Wuts, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d John Wiley and Sons, New York (1991), the disclosure of which is hereby incorporated by reference. Any thiol protecting group known in the art can be used. Examples of thiol protecting groups include, but are not limited to, the following: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, and triphenylmethyl.
Exemplary protecting groups for hydrazine bonding units are hydrazones which can be aldehyde or ketone hydrazones having substituents selected from hydrogen, alkyl, aryl and heterocycle. Particularly preferred hydrazones are described in co-pending U.S. Ser. No. 08/476,296 the disclosure of which is herein incorporated by reference in its entirety.
The hydrazine bonding unit when bound to a metal radionuclide is termed a hydrazido, or diazenido group and serves as the point of attachment of the radionuclide to the remainder of the radiopharmaceutical. A diazenido group can be either terminal (only one atom of the group is bound to the radionuclide) or chelating. In order to have a chelating diazenido group at least one other atom of the group must also be bound to the radionuclide. The atoms bound to the metal are termed donor atoms.
Chelators for 111In and 86Y are selected from cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA, and 6,6xe2x80x3-bis[N,N,Nxe2x80x3,Nxe2x80x3-tetra(carboxymethyl)aminomethyl)-4xe2x80x2-(3-amino-4-methoxyphenyl)-2,2xe2x80x2:6xe2x80x2,2xe2x80x3-terpyridine. Procedures for synthesizing these chelators that are not commercially available can be found in Brechbiel, M. and Gansow, O., J. Chem. Soc. Perkin Trans. 1992, 1, 1175; Brechbiel, M. and Gansow, O., Bioconjugate Chem. 1991, 2, 187; Deshpande, S., et. al., J. Nucl. Med. 1990, 31, 473; Kruper, J., U.S. Pat. No. 5,064,956, and Toner, J., U.S. Pat. No. 4,859,777, the disclosures of which are hereby incorporated by reference in their entirety.
The coordination sphere of metal ion includes all the ligands or groups bound to the metal. For a transition metal radionuclide to be stable it typically has a coordination number (number of donor atoms) comprised of an integer greater than or equal to 4 and less than or equal to 8; that is there are 4 to 8 atoms bound to the metal and it is said to have a complete coordination sphere. The requisite coordination number for a stable radionuclide complex is determined by the identity of the radionuclide, its oxidation state, and the type of donor atoms. If the chelator or bonding unit does not provide all of the atoms necessary to stabilize the metal radionuclide by completing its coordination sphere, the coordination sphere is completed by donor atoms from other ligands, termed ancillary or co-ligands, which can also be either terminal or chelating.
A large number of ligands can serve as ancillary or co-ligands, the choice of which is determined by a variety of considerations such as the ease of synthesis of the radiopharmaceutical, the chemical and physical properties of the ancillary ligand, the rate of formation, the yield, and the number of isomeric forms of the resulting radiopharmaceuticals, the ability to administer said ancillary or co-ligand to a patient without adverse physiological consequences to said patient, and the compatibility of the ligand in a lyophilized kit formulation. The charge and lipophilicity of the ancillary ligand will effect the charge and lipophilicity of the radiopharmaceuticals. For example, the use of 4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticals with an additional two anionic groups because the sulfonate groups will be anionic under physiological conditions. The use of N-alkyl substituted 3,4-hydroxypyridinones results in radiopharmaceuticals with avarying degrees of lipophilicity depending on the size of the alkyl substituents.
Preferred technetium radiopharmaceuticals of the present invention are comprised of a hydrazido or diazenido bonding unit and an ancillary ligand, AL1, or a bonding unit and two types of ancillary AL1 and AL2, or a tetradentate chelator comprised of two nitrogen and two sulfur atoms. Ancillary ligands AL1 are comprised of two or more hard donor atoms such as oxygen and amine nitrogen (sp3 hybridized). The donor atoms occupy at least two of the sites in the coordination sphere of the radionuclide metal; the ancillary ligand AL1 serves as one of the three ligands in the ternary ligand system. Examples of ancillary ligands AL1 include but are not limited to dioxygen ligands and functionalized aminocarboxylates. A large number of such ligands are available from commercial sources.
Ancillary dioxygen ligands include ligands that coordinate to the metal ion through at least two oxygen donor atoms. Examples include but are not limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate, lactate, tartrate, mannitol, glucarate, maltol, Kojic acid, 2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzene disulfonate, or substituted or unsubstituted 1,2 or 3,4 hydroxypyridinones. (The names for the ligands in these examples refer to either the protonated or non-protonated forms of the ligands.)
Functionalized aminocarboxylates include ligands that have a combination of amine nitrogen and oxygen donor atoms. Examples include but are not limited to: iminodiacetic acid, 2,3-diaminopropionic acid, nitrilotriacetic acid, N,Nxe2x80x2-ethylenediamine diacetic acid, N,N,Nxe2x80x2-ethylenediamine triacetic acid, hydroxyethylethylenediamine triacetic acid, and N,Nxe2x80x2-ethylenediamine bis-hydroxyphenylglycine. (The names for the ligands in these examples refer to either the protonated or non-protonated forms of the ligands.)
A series of functionalized aminocarboxylates are disclosed by Bridger et. al. in U.S. Pat. No. 5,350,837, herein incorporated by reference, that result in improved rates of formation of technetium labeled hydrazino modified proteins. We have determined that certain of these aminocarboxylates result in improved yields of the radiopharmaceuticals of the present invention. The preferred ancillary ligands AL1 functionalized aminocarboxylates that are derivatives of glycine; the most preferred is tricine (tris(hydroxymethyl)methylglycine).
The most preferred technetium radiopharmaceuticals of the present invention are comprised of a hydrazido or diazenido bonding unit and two types of ancillary designated AL1 and AL2, or a diaminedithiol chelator. The second type of ancillary ligands AL2 are comprised of one or more soft donor atoms selected from the group: phosphine phosphorus, arsine arsenic, imine nitrogen (sp2 hybridized), sulfur (sp2 hybridized) and carbon (sp hybridized); atoms which have p-acid character. Ligands AL2 can be monodentate, bidentate or tridentate, the denticity is defined by the number of donor atoms in the ligand. One of the two donor atoms in a bidentate ligand and one of the three donor atoms in a tridentate ligand must be a soft donor atom. We have disclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. No. 60/013360 and U.S. Ser. No. 08/646,886, the disclosures of which are herein incorporated by reference in their entirety, that radiopharmaceuticals comprised of one or more ancillary or co-ligands AL2 are more stable compared to radiopharmaceuticals that are not comprised of one or more ancillary ligands, AL2; that is, they have a minimal number of isomeric forms, the relative ratios of which do not change significantly with time, and that remain substantially intact upon dilution.
The ligands AL2 that are comprised of phosphine or arsine donor atoms are trisubstituted phosphines, trisubstituted arsines, tetrasubstituted diphosphines and tetrasubstituted diarsines. The ligands AL2 that are comprised of imine nitrogen are unsaturated or aromatic nitrogen-containing, 5 or 6-membered heterocycles. The ligands that are comprised of sulfur (sp2 hybridized) donor atoms are thiocarbonyls, comprised of the moiety Cxe2x95x90S. The ligands comprised of carbon (sp hybridized) donor atoms are isonitriles, comprised of the moiety CNR, where R is an organic radical. A large number of such ligands are available from commercial sources. Isonitriles can be synthesized as described in European Patent 0107734 and in U.S. Pat. No. 4,988,827, herein incorporated by reference.
Preferred ancillary ligands AL2 are trisubstituted phosphines and unsaturated or aromatic 5 or 6 membered heterocycles. The most preferred ancillary ligands AL2 are trisubstituted phosphines and unsaturated 5 membered heterocycles.
The ancillary ligands AL2 may be substituted with alkyl, aryl, alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may or may not bear functional groups comprised of heteroatoms such as oxygen, nitrogen, phosphorus or sulfur. Examples of such functional groups include but are not limited to: hydroxyl, carboxyl, carboxamide, nitro, ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, and phosphonamide. The functional groups may be chosen to alter the lipophilicity and water solubility of the ligands which may affect the biological properties of the radiopharmaceuticals, such as altering the distribution into non-target tissues, cells or fluids, and the mechanism and rate of elimination from the body.
Chelators or bonding moieties for therapeutic radiopharmaceuticals are selected to form stable complexes with the radioisotopes that have alpha particle, beta particle, Auger or Coster-Kronig electron emissions, such as 186Re, 188Re, 153Sm, 166Ho, 177Lu, 149Pm, 90Y, 212Bi, 103Pd, 109Pd, 159Gd, 140La, 198Au, 199Au, 169Yb, 175Yb, 165Dy, 166Dy, 67Cu, 105Rh, 111Ag, and 192Ir. Chelators for rhenium, copper, palladium, platinum, iridium, rhodium, silver and gold isotopes are selected from diaminedithiols, monoamine-monoamidedithiols, triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Chelators for yttrium, bismuth, and the lanthanide isotopes are selected from cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA, and 6,6xe2x80x3-bis[N,N,Nxe2x80x3,Nxe2x80x3-tetra(carboxymethyl)aminomethyl)-4xe2x80x2-(3-amino-4-methoxyphenyl)-2,2xe2x80x2:6xe2x80x2,2xe2x80x3-terpyridine.
Chelators for magnetic resonance imaging contrast agents are selected to form stable complexes with paramagnetic metal ions, such as Gd(III), Dy(III), Fe(III), and Mn(II), are selected from cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA, alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid, 2-benzyl-6-methyl-DTPA, and 6,6xe2x80x3-bis[N,N,Nxe2x80x3,Nxe2x80x3-tetra(carboxymethyl)aminomethyl)-4xe2x80x2-(3-amino-4-methoxyphenyl)-2,2xe2x80x2:6xe2x80x2,2-terpyridine.
The technetium and rhenium radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be easily prepared by admixing a salt of a radionuclide, a reagent of the present invention, an ancillary ligand AL1, an ancillary ligand AL2, and a reducing agent, in an aqueous solution at temperatures from 0 to 100xc2x0 C. The technetium and rhenium radiopharmaceuticals of the present invention comprised of a tetradentate chelator having two nitrogen and two sulfur atoms can be easily prepared by admixing a salt of a radionuclide, a reagent of the present invention, and a reducing agent, in an aqueous solution at temperatures from 0 to 100xc2x0 C.
When the bonding unit in the reagent of the present invention is present as a hydrazone group, then it must first be converted to a hydrazine, which may or may not be protonated, prior to complexation with the metal radionuclide. The conversion of the hydrazone group to the hydrazine can occur either prior to reaction with the radionuclide, in which case the radionuclide and the ancillary or co-ligand or ligands are combined not with the reagent but with a hydrolyzed form of the reagent bearing the chelator or bonding unit, or in the presence of the radionuclide in which case the reagent itself is combined with the radionuclide and the ancillary or co-ligand or ligands. In the latter case, the pH of the reaction mixture must be neutral or acidic.
Alternatively, the radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be prepared by first admixing a salt of a radionuclide, an ancillary ligand AL1, and a reducing agent in an aqueous solution at temperatures from 0 to 100xc2x0 C. to form an intermediate radionuclide complex with the ancillary ligand AL1 then adding a reagent of the present invention and an ancillary ligand AL2 and reacting further at temperatures from 0 to 100xc2x0 C.
Alternatively, the radiopharmaceuticals of the present invention comprised of a hydrazido or diazenido bonding unit can be prepared by first admixing a salt of a radionuclide, an ancillary ligand AL1, a reagent of the present invention, and a reducing agent in an aqueous solution at temperatures from 0 to 100xc2x0 C. to form an intermediate radionuclide complex, and then adding an ancillary ligand AL2 and reacting further at temperatures from 0 to 100xc2x0 C.
The technetium and rhenium radionuclides are preferably in the chemical form of pertechnetate or perrhenate and a pharmaceutically acceptable cation. The pertechnetate salt form is preferably sodium pertechnetate such as obtained from commercial Tc-99m generators. The amount of pertechnetate used to prepare the radiopharmaceuticals of the present invention can range from 0.1 mCi to 1 Ci, or more preferably from 1 to 200 mCi.
The amount of the reagent of the present invention used to prepare the technetium and rhenium radiopharmaceuticals of the present invention can range from 0.01 xcexcg to 10 mg, or more preferably from 0.5 xcexcg to 200 xcexcg. The amount used will be dictated by the amounts of the other reactants and the identity of the radiopharmaceuticals of the present invention to be prepared.
The amounts of the ancillary ligands AL1 used can range from 0.1 mg to 1 g, or more preferably from 1 mg to 100 mg. The exact amount for a particular radiopharmaceutical is a function of identity of the radiopharmaceuticals of the present invention to be prepared, the procedure used and the amounts and identities of the other reactants. Too large an amount of AL1 will result in the formation of by-products comprised of technetium labeled AL1 without a biologically active molecule or by-products comprised of technetium labeled biologically active molecules with the ancillary ligand AL1 but without the ancillary ligand AL2. Too small an amount of AL1 will result in other by-products such as technetium labeled biologically active molecules with the ancillary ligand AL2 but without the ancillary ligand AL1, or reduced hydrolyzed technetium, or technetium colloid.
The amounts of the ancillary ligands AL2 used can range from 0.001 mg to 1 g, or more preferably from 0.01 mg to 10 mg. The exact amount for a particular radiopharmaceutical is a function of the identity of the radiopharmaceuticals of the present invention to be prepared, the procedure used and the amounts and identities of the other reactants. Too large an amount of AL2 will result in the formation of by-products comprised of technetium labeled AL2 without a biologically active molecule or by-products comprised of technetium labeled biologically active molecules with the ancillary ligand AL2 but without the ancillary ligand AL1. If the reagent bears one or more substituents that are comprised of a soft donor atom, as defined above, at least a ten-fold molar excess of the ancillary ligand AL2 to the reagent of formula 2 is required to prevent the substituent from interfering with the coordination of the ancillary ligand AL2 to the metal radionuclide.
Suitable reducing agents for the synthesis of the radiopharmaceuticals of the present invention include stannous salts, dithionite or bisulfite salts, borohydride salts, and formamidinesulfinic acid, wherein the salts are of any pharmaceutically acceptable form. The preferred reducing agent is a stannous salt. The amount of a reducing agent used can range from 0.001 mg to 10 mg, or more preferably from 0.005 mg to 1 mg.
The specific structure of a radiopharmaceutical of the present invention comprised of a hydrazido or diazenido bonding unit will depend on the identity of the reagent of the present invention used, the identity of any ancillary ligand AL1, the identity of any ancillary ligand AL2, and the identity of the radionuclide. Radiopharmaceuticals comprised of a hydrazido or diazenido bonding unit synthesized using concentrations of reagents of  less than 100 xcexcg/mL, will be comprised of one hydrazido or diazenido group. Those synthesized using  greater than 1 mg/mL concentrations will be comprised of two hydrazido or diazenido groups from two reagent molecules. For most applications, only a limited amount of the biologically active molecule can be injected and not result in undesired side-effects, such as chemical toxicity, interference with a biological process or an altered biodistribution of the radiopharmaceutical. Therefore, the radiopharmaceuticals which require higher concentrations of the reagents comprised in part of the biologically active molecule, will have to be diluted or purified after synthesis to avoid such side-effects.
The identities and amounts used of the ancillary ligands AL1 and AL2 will determine the values of the variables y and z. The values of y and z can independently be an integer from 1 to 2. In combination, the values of y and z will result in a technetium coordination sphere that is made up of at least five and no more than seven donor atoms. For monodentate ancillary ligands AL2, z can be an integer from 1 to 2; for bidentate or tridentate ancillary ligands AL2, z is 1. The preferred combination for monodentate ligands is y equal to 1 or 2 and z equal to 1. The preferred combination for bidentate or tridentate ligands is y equal to 1 and z equal to 1.
The indium, copper, gallium, silver, palladium, rhodium, gold, platinum, bismuth, yttrium and lanthanide radiopharmaceuticals of the present invention can be easily prepared by admixing a salt of a radionuclide and a reagent of the present invention, in an aqueous solution at temperatures from 0 to 100xc2x0 C. These radionuclides are typically obtained as a dilute aqueous solution in a mineral acid, such as hydrochloric, nitric or sulfuric acid. The radionuclides are combined with from one to about one thousand equivalents of the reagents of the present invention dissolved in aqueous solution. A buffer is typically used to maintain the pH of the reaction mixture between 3 and 10.
The gadolinium, dysprosium, iron and manganese metallopharmaceuticals of the present invention can be easily prepared by admixing a salt of the paramagnetic metal ion and a reagent of the present invention, in an aqueous solution at temperatures from 0 to 100xc2x0 C. These paramagnetic metal ions are typically obtained as a dilute aqueous solution in a mineral acid, such as hydrochloric, nitric or sulfuric acid. The paramagnetic metal ions are combined with from one to about one thousand equivalents of the reagents of the present invention dissolved in aqueous solution. A buffer is typically used to maintain the pH of the reaction mixture between 3 and 10.
The total time of preparation will vary depending on the identity of the metal ion, the identities and amounts of the reactants and the procedure used for the preparation. The preparations may be complete, resulting in  greater than 80% yield of the radiopharmaceutical, in 1 minute or may require more time. If higher purity metallopharmaceuticals are needed or desired, the products can be purified by any of a number of techniques well known to those skilled in the art such as liquid chromatography, solid phase extraction, solvent extraction, dialysis or ultrafiltration.
Buffers useful in the preparation of metallopharmaceuticals and in diagnostic kits useful for the preparation of said radiopharmaceuticals include but are not limited to phosphate, citrate, sulfosalicylate, and acetate. A more complete list can be found in the United States Pharmacopeia.
Lyophilization aids useful in the preparation of diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to mannitol, lactose, sorbitol, dextran, Ficoll, and polyvinylpyrrolidine(PVP).
Stabilization aids useful in the preparation of metallopharmaceuticals and in diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid, and inositol.
Solubilization aids useful in the preparation of metallopharmaceuticals and in diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monoloeate, polysorbates, poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers (Pluronics) and lecithin. Preferred solubilizing aids are polyethylene glycol, and Pluronics.
Bacteriostats useful in the preparation of metallopharmaceuticals and in diagnostic kits useful for the preparation of radiopharmaceuticals include but are not limited to benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl, propyl or butyl paraben.
A component in a diagnostic kit can also serve more than one function. A reducing agent can also serve as a stabilization aid, a buffer can also serve as a transfer ligand, a lyophilization aid can also serve as a transfer, ancillary or co-ligand and so forth.
The diagnostic radiopharmaceuticals are administered by intravenous injection, usually in saline solution, at a dose of 1 to 100 mCi per 70 kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging is performed using known procedures.
The therapeutic radiopharmaceuticals are administered by intravenous injection, usually in saline solution, at a dose of 0.1 to 100 mCi per 70 kg body weight, or preferably at a dose of 0.5 to 5 mCi per 70 kg body weight.
The magnetic resonance imaging contrast agents of the present invention may be used in a similar manner as other MRI agents as described in U.S. Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt et al., Magn. Reson. Med., 1986, 3, 808; Runge et al., Radiology, 1988, 166, 835; and Bousquet et al., Radiology, 1988, 166, 693. Generally, sterile aqueous solutions of the contrast agents are administered to a patient intravenously in dosages ranging from 0.01 to 1.0 mmoles per kg body weight.
For use as X-ray contrast agents, the compositions of the present invention should generally have a heavy atom concentration of 1 mM to 5 M, preferably 0.1 M to 2 M. Dosages, administered by intravenous injection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg, preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using known techniques, preferably X-ray computed tomography.
The ultrasound contrast agents of the present invention are administered by intravenous injection in an amount of 10 to 30 xcexcL of the echogenic gas per kg body weight or by infusion at a rate of approximately 3 xcexcL/kg/min. Imaging is performed using known techniques of sonography.